The gonadotropins form a family of structurally related glycoprotein hormones. Typical members include chorionic gonadotropin (CG), follicle stimulating hormone (FSH; follitropin), luteinizing hormone (LH; lutropin) and thyroid stimulating hormone (TSH; thyrotropin). FSH, LH and TSH are present in most vertebrate species and are synthesized and secreted by the pituitary. CG has so far been found only in primates, including humans, and in horses and is synthesized by placental tissue. FSH and LH are the pituitary hormones essential for follicular maturation and luteinization in the female and for testis maturation and spermatogenesis in the male. Purified FSH administered alone or in combination with semipurified human menopausal gonadotropins containing a mixture of FSH and LH has been used, among others, to stimulate the development of ovarian follicles, as is required for assisted reproduction techniques, such as the IVF (in vitro fertilization) method. Human FSH, partially purified from urine is also used clinically to stimulate follicular maturation in anovulatory women with chronic anovulatory syndrome or luteal phase deficiency. In males a combination of FSH and LH have been used in a variety of conditions related to male infertility.
In recent years very pure preparations, of the gonadotropins have become available through the use of recombinant DNA technology (see for instance Boime et al., Seminars in Reproductive Endocrinology 10, 45-50, 1992: "Expression of recombinant human FSH, LH and CG in mammalian cells"). The recombinant gonadotropins are of constant quality i.e. have reproducible biochemical and biological properties. Genomic and cDNA clones have been prepared for all subunits and their primary structure has been resolved. Moreover, Chinese Hamster Ovary (CHO) cells have been transfected with human gonadotropin subunit genes and these cells are shown to be capable of secreting intact dimers (e.g. Keene et al (1989), J.Biol.Chem., 264, 4769-4775; Van Wezenbeek et al (1990), in From clone to Clinic (eds Crommelin D. J. A. and Schellekens H.), 245-251). It has been demonstrated that the biochemical and biological characteristics of e.g. recombinant FSH are almost identical to those of natural FSH (Mannaerts et al (1991), Endocrinology, 129, 2623-2630). Moreover, pregnancies were achieved after controlled ovarian superovulation using recombinant FSH (Germond et al (1992), Lancet, 339 ,1170; Devroey et al (1992), Lancet, 339, 1170-1171).
Structurally the gonadotropins are heterodimers composed of two dissimilar subunits, named .alpha. and .beta., which are associated by non-covalent bonds. Within a species, the .alpha.-subunit is essentially identical for each member of the gonadotropin family; it is also highly conserved from species to species. The .beta.-subunits are different for each member, i.e. CG, FSH, TSH and LH, but show considerable homology in structure. Furthermore, also the .beta. subunits are highly conserved from species to species. In humans, the .alpha. subunit consists of 92 amino acid residues, whilst the .beta. subunit varies in size for each member: 111 residues in hFSH, 121 residues in hLH, 118 residues in hTSH and 145 residues in hCG (Combamous, Y. (1992), Endocrine Reviews, 13, 670-691; Lustbader, J. W. et al. (1993), Endocrine Reviews, 14, 291-311). The .beta. subunit of hCG is substantially larger than the other .beta. subunits in that it contains approximately 34 additional amino acids at the C-terminus referred to herein as the carboxy terminal protein (CTP).
Relatively pure gonadotropin preparations are commercially available. For example, compositions containing naturally derived human menopausal gonadotropin (hMG), with FSH and LH activities in a ratio of approximately 1:1, and naturally derived human chorionic gonadotropin (hCG) are available, for example, as freeze-dried preparations under the trade names Humegon.RTM. and Pregnyl.RTM., respectively, from N. V. Organon, Oss, The Netherlands. A freeze-dried recombinant human FSH (recFSH) preparation is, for example, available under the trade name Puregon.RTM. from the same company. The recombinant FSH is likewise in use for ovulation induction and for controlled ovarian hyperstimulation.
The stability of proteins in aqueous formulations is generally a problem in pharmaceutical industry. Likewise the stability of aqueous solutions of the gonadotropins is insufficient to allow storage for longer times. This is especially true for preparations containing the very pure gonadotropins, prepared using recombinant DNA methods, in relatively dilute solutions . Usually therefore those preparations are stored in a dry form, as is obtained after lyophilization. A stabilized gonadotropin containing lyophilized pharmaceutical formulation is disclosed in European Patent No. 448,146 (Akzo N. V.). These preparations contain organic carboxylic acids, particularly citric acid, and optionally a non-reducing sugar such as sucrose. Another solid gonadotropin containing pharmaceutical composition comprising sucrose as a stabilizer is disclosed in the International Patent Application WO 93/11788 (Applied Research Systems ARS Holding N. V.).
Although these freeze-dried preparations are stable enough to guarantee sufficient shelf-lifes, they have the disadvantage that prior to administration reconstitution is necessary. The patient therefore necessarily has to reconstitute the dried glycoprotein in a solvent before use, which is a disadvantage and an inconvenience to the patient. In addition, the solvent must be provided together with the freeze-dried preparation of the gonadotropin.
For a patient, who needs injections of a gonadotropin at regular times, for instance a patient receiving a daily dose of recFSH for ovulation induction, it would be of importance that the gonadotropin formulation is easy to handle, to dose and to inject. The reconstitution of a freeze-dried gonadotropin preparation demands prudence and carefulness and should be avoided if possible. It would facilitate the use of gonadotropins, if these glycoproteins could be produced and distributed as a stable solution to the patient, who could inject the medicament directly without reconstitution. In addition, a freeze-drying process is a costly and time consuming process step, and it would be an advantage if this step could be avoided when preparing a gonadotropin formulation.
A need exists therefore in a ready-for-use injection preparation, having a sufficient stability to guarantee a reasonable shelf-life.
In WO 93/22335 (COR Therapeutics Inc.) storage stable liquid compositions of substantially pure polypeptides are disclosed, which are prepared by dissolving the polypeptide in a citrate buffer of pH 5.0 to 5.5. Liquid formulations containing the gonadotropin recombinant-hCG stabilized with a non reducing sugar, preferably mannitol, in an aqueous solution in a phosphate buffer at pH 7, are disclosed in WO 96/29095 (Applied Research Systems ARS Holding N. V.).
Solutions comprising gonadotropins and a polycarboxylic acid salt are known from European Patent 448,146 (Akzo N. V.). These solutions, containing for instance citric acid, are described for preparing stabilized lyophilised gonadotropin formulations.
On storage of such solutions per se for longer times (months at room temperature) the gonadotropins are insufficiently stable.